The polymerase chain reaction (PCR) is based on repeated cycles of denaturation of double stranded DNA, followed by oligonucleotide primer annealing to the DNA template, and primer extension by a DNA polymerase (eg see Mullis et al U.S. Pat. Nos. 4,683,195, 4,683,202 and 4,800,159). The oligonucleotide primers used in PCR are designed to anneal to opposite strands of the DNA, and are positioned so that the DNA polymerase-catalysed extension product of one primer can serve as a template strand for the other primer. The PCR amplification method results in the exponential increase of discrete DNA the length of which is defined by the 5′ ends of the oligonucleotide primers.
In its standard application, primers are chosen that match sequences within the target genome and flank the region of DNA that is to be amplified. Additionally a number of variations of PCR have been described in which linkers are ligated onto the ends of DNA fragments and sequences in the linkers are then used for priming DNA amplification. Ligation-mediated PCR was first applied to DNA footprinting and sequencing reactions where the DNA ends were generated by DNaseI digestion or chemical cleavage (Mueller and Wold, 1989 and Pfeifer et al. 1989) and extended to ends formed by restriction digestion (Steigerwald et al. 1990). Specificity is achieved by combining primers to a specific target region with primers targeted to the added linker. Variations of the technique have found a range of uses in genome sequencing and DNA methylation analysis, chromosome walking, identifying sites of chromosome integration or recombination, studying mutation breakpoints and mRNA termini. Ligation-mediated PCR can also be used for whole genome amplification, where the entire population of molecules with ligated ends is amplified (Schumaker et al., 2006).
Despite the wide-ranging usefulness of ligation-mediated PCR, there is an ongoing need for improved PCR-based methodologies that allow for selective amplification of DNAs having 3′ ends and that provide advantages of simplicity, specificity and expediency.